*2.13. Artesunate Susceptibility in the Presence of Inhibitors*

The susceptibility of K133WT and K133AS-R parasites towards artesunate was determined in the presence of the AQP1 inhibitor (Tocris Biosciences, Bristol, UK) and ABC transporter modulator, verapamil (Sigma, St. Louis, MO, USA). At the promastigote stage, both K133WT and K133AS-R isolates (1 × 10<sup>5</sup> ) were seeded into a 96-well plate with various concentrations of artesunate drug (1–650 µM) alone or in the presence of 40 µM of AQP1 inhibitor or 8 µM of verapamil and incubated at 25 ◦C. After 72 h of incubation, 50 µL of Resazurin (Sigma Aldrich, St. Louis, MO, USA) (0.0125% (*w*/*v*) in Phosphate Buffered Saline (PBS) were added to each well and the plates were further incubated for 18 h. Fluorescence was measured at an excitation wavelength of 550 nm and emission wavelength of 590 nm on an Infinite M200 multimode reader (Tecan, Switzerland) to determine cell viability. Sigmoidal regression analysis was used to calculate IC<sup>50</sup> [24].

At the amastigote stage, the mice PECs were infected with late log-phase promastigotes of K133WT or K133AS-R at a ratio of 10 parasites: 1 macrophage, plated into 8-well chamber slides and incubated for 16 h at 37 ◦C in 5% CO2. Non-internalized promastigotes were washed off and infected macrophages were further incubated with various dilutions of artesunate drug (13, 26, 52, 104, 208, and 260 µM) with or without AQP1 inhibitor (Tocris Biosciences, Bristol, UK) (40 µM) or verapamil (8 µM). The inhibitor/modulator alone at the tried concentration was not lethal to either K133WT/K133AS-R isolates or host macrophages. Then, 48 h later, the slides were fixed and stained using Diff-Quik solutions. Macrophages were then examined for intracellular amastigotes at 1000 × magnification. The number of *L. donovani* amastigotes per 100 macrophages was counted and the survival rate of parasites relative to untreated macrophages was calculated to determine the IC<sup>50</sup> value [24].
